Delphine Grivet

Gene movement and genetic association with regional climate gradients in California valley oak (Quercus lobata Née) in the face of climate change

Rapid climate change jeopardizes tree populations by shifting current climate zones. To avoid extinction, tree populations must tolerate, adapt, or migrate. Here we investigate geographic patterns of genetic variation in valley oak, Quercus lobata Née, to assess how underlying genetic structure of populations might influence this species’ ability to survive climate change. First, to understand how genetic lineages shape spatial genetic patterns, we examine historical patterns of colonization. Second, we examine the correlation between multivariate nuclear genetic variation and climatic variation. Third, to illustrate how geographic genetic variation could interact with regional patterns of 21st Century climate change, we produce region‐specific bioclimatic distributions of valley oak using Maximum Entropy (MAXENT) models based on downscaled historical (1971–2000) and future (2070–2100) climate grids. Future climatologies are based on a moderate‐high (A2) carbon emission scenario and two different global climate models. Chloroplast markers indicate historical range‐wide connectivity via colonization, especially in the north. Multivariate nuclear genotypes show a strong association with climate variation that provides opportunity for local adaptation to the conditions within their climatic envelope. Comparison of regional current and projected patterns of climate suitability indicates that valley oaks grow in distinctly different climate conditions in different parts of their range. Our models predict widely different regional outcomes from local displacement of a few kilometres to hundreds of kilometres. We conclude that the relative importance of migration, adaptation, and tolerance are likely to vary widely for populations among regions, and that late 21st Century conditions could lead to regional extinctions.

Phylogeography of the common ivy (Hedera sp.) in Europe: genetic differentiation through space and time

We studied the phylogeography of ivy (Hedera sp.), a liana widespread in Europe, throughout its natural range. The populations sampled belong to four closely related species differing by ploidy levels and morphological characters. Chloroplast (cp) markers were used and 13 haplotypes were detected, usually shared across species, contrary to ribosomal internal transcribed spacer (ITS) variants. We demonstrated the existence of a strong overall cpDNA phylogeographical structure. Several methods of data analysis were conducted to describe how this structure and the genetic diversity change through space and time. Southern populations, especially those from Spain, are the most divergent. Pairwise estimates of differentiation point to isolation by distance, and the existence of a latitudinal gradient of divergence was demonstrated using a regression procedure. Similarly, latitudinal differences in haplotype richness and diversity exist, as shown by population permutations (‘differentiation through space’). Finally, we measured differentiation by taking into account successive levels of divergence between haplotypes (‘differentiation through time’). Genetic differentiation turns out to be much greater when differences between closely related haplotypes are not considered. Further, these results indicate that the phylogeographical structure is essentially due to the relative distribution of the most similar haplotypes. Diversity decreases from south to north, whereas haplotype frequencies change longitudinally. It appears that Hedera survived in Spanish and Balkan refugia during the last ice age. A third refugium must have been present in the Alps or in Italy. During the northward expansion, the decrease in overall diversity was attenuated by some mixing of lineages at intermediate latitudes, resulting in comparatively higher levels of differentiation in the south.

Chloroplast DNA phylogeography of the hornbeam in Europe: Evidence for a bottleneck at the outset of postglacial colonization

The hornbeam, a shade tolerant tree species,has recolonised Europe very late during theHolocene. In order to investigate whether thispostglacial recolonisation had led to a loss ofchloroplast (cp) DNA diversity, as alreadydescribed for other tree species, andespecially the beech, another late-successionalspecies, we have studied the phylogeography ofthe hornbeam using cpDNA. Three types of cpDNAmarkers were used (PCR-RFLP, microsatellites,sequences) to analyse 36 European populationsof C. betulus and five populations ofC. orientalis. Six haplotypes specific toC. betulus were detected, one of themcompletely fixed in all populations fromnorthern and western Europe, whereas the otherswere restricted to the eastern Europeancountries and to southern Italy. A remarkablyhigh value of differentiation among populationswas found (GST = 0.972). Twodifferent haplotypes specific to C.orientalis were detected, suggesting noongoing gene flow between these two species,and similar refugia in Italy and in the Balkansfor both Carpinus species. Pollen dataare congruent with genetic data: during thelast ice age, the hornbeam was restricted tothe Balkans and to southern Italy andrecolonised Europe northwards and westwards.Conservation priorities should target severalpopulations from these regions that arehotspots of diversity and that represent asmall part of the range of the species.

Molecular footprints of local adaptation in two Mediterranean conifers

This study combines neutrality tests and environmental correlations to identify nonneutral patterns of evolution in candidate genes related to drought stress in two closely related Mediterranean conifers, Pinus pinaster Ait. and P. halepensis Mill. Based on previous studies, we selected twelve amplicons covering six candidate genes that were sequenced in a large sample spanning the full range of these two species. Neutrality tests relatively robust to demography (DHEW compound test and maximum likelihood multilocus Hudson-Kreitman-Aguadé test) were used to detect selection events at different temporal scales. Environmental associations between variation at candidate genes and climatic variables were also examined. These combined approaches detected distinct genes that may be targeted by selection, most of them specific to only one of the two conifers, despite their recent divergence (<10 Ma). An exception was 4-coumarate: CoA ligase, a gene involved in the production of various important secondary products that appeared to play a role in local adaptation processes of both pines. Another remarkable result was that all significant environmental correlations involved temperature indices, highlighting the importance of this climatic factor as a selective driver on Mediterranean pines. The ability to detect natural selection at the DNA sequence level depends on the nature and the strength of the selection events, on the timescale at which they occurred, and on the sensitivity of the methods to other evolutionary forces that can mimic selection (e.g., demography and population structure). Using complementary approaches can help to capture different aspects of the evolutionary processes that govern molecular variation at both intra- and interspecific levels.

Development and implementation of a highly- multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine

BackgroundSingle nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe.ResultsWe designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 in vitro SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 in silico SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for in silico and in vitro SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species.ConclusionsOur results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.

The Strait of Gibraltar as a major biogeographic barrier in Mediterranean conifers: a comparative phylogeographic survey

The Strait of Gibraltar (SG) is reputed for being both a bridge and a geographic barrier to biological exchanges between Europe and Africa. Major genetic breaks associated with this strait have been identified in various taxa, but it is unknown whether these disjunctions have been produced simultaneously or by independent biogeographic processes. Here, the genetic structure of five conifers distributed on both sides of the SG was investigated using mitochondrial (nad1 b/c, nad5‐1, nad5‐4 and nad7‐1) and chloroplast (Pt1254, Pt15169, Pt30204, Pt36480, Pt71936 and Pt87268) DNA markers. The distribution of genetic variation was partially congruent between types of markers within the same species. Across taxa, there was a significant overlapping between the SG and the genetic breaks detected, especially for the four Tertiary species surveyed (Abies pinsapo complex, Pinus nigra, Pinus pinaster and Taxus baccata). For most of these taxa, the divergence of populations across the SG could date back to long before the Pleistocene glaciations. However, their strongly different cpDNA GST and RST values point out that they have had dissimilar population histories, which might include contrasting amounts of pollen‐driven gene flow since their initial establishment in the region. The fifth species, Pinus halepensis, was genetically depauperated and homogenous on both sides of the SG. A further analysis of nuclear DNA sequences with coalescent‐based isolation with migration models suggests a Pleistocene divergence of P. halepensis populations across the SG, which is in sharp contrast with the pre‐Pleistocene divergence dates obtained for P. pinaster. Altogether, these results indicate that the genetic breaks observed across this putative biogeographical barrier have been produced by independent evolutionary processes related to the biological history of each individual species instead of a common vicariant phenomenon.

Molecular Proxies for Climate Maladaptation in a Long-Lived Tree (Pinus Pinaster Aiton, Pinaceae)

Understanding adaptive genetic responses to climate change is a main challenge for preserving biological diversity. Successful predictive models for climate-driven range shifts of species depend on the integration of information on adaptation, including that derived from genomic studies. Long-lived forest trees can experience substantial environmental change across generations, which results in a much more prominent adaptation lag than in annual species. Here, we show that candidate-gene SNPs (single nucleotide polymorphisms) can be used as predictors of maladaptation to climate in maritime pine (Pinus pinaster Aiton), an outcrossing long-lived keystone tree. A set of 18 SNPs potentially associated with climate, 5 of them involving amino acid-changing variants, were retained after performing logistic regression, latent factor mixed models, and Bayesian analyses of SNP–climate correlations. These relationships identified temperature as an important adaptive driver in maritime pine and highlighted that selective forces are operating differentially in geographically discrete gene pools. The frequency of the locally advantageous alleles at these selected loci was strongly correlated with survival in a common garden under extreme (hot and dry) climate conditions, which suggests that candidate-gene SNPs can be used to forecast the likely destiny of natural forest ecosystems under climate change scenarios. Differential levels of forest decline are anticipated for distinct maritime pine gene pools. Geographically defined molecular proxies for climate adaptation will thus critically enhance the predictive power of range-shift models and help establish mitigation measures for long-lived keystone forest trees in the face of impending climate change.

First insights into the transcriptome and development of new genomic tools of a widespread circum-Mediterranean tree species, Pinus halepensis Mill

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next‐generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA‐Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene‐based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48 629 contigs and covered about 54.6 Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein‐coding sequences resulted in the detection of 34 014 SNPs across species, with a Ka/Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione‐s‐transferase, a cellulose synthase and a cobra‐like protein. A large number of new markers (3334 amplifiable SSRs and 28 236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384‐SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next‐generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.

PSA: software for parental structure analysis of seed or seedling patches

Parental structure analysis (PSA) is a computer program to analyse separate contributions of paternal and maternal parents to postdispersal plant offspring. The program provides joint estimates of maternal, paternal and cross‐parental correlations within and among a set of predefined groups of seeds or seedlings, as well as derivative estimates of effective parental numbers. PSA utilizes data sets that distinguish between maternal and paternal contributions to the genotype of each offspring in the sample, but does not require parental samples per se. The approach requires assay of codominant diploid markers from both seed coat (maternally inherited) and seedling/embryo (biparentally inherited) tissues for each offspring. A simulation analysis of PSAs performance shows that it provides fairly accurate parental correlation estimates from affordable sampling effort. PSA should be of interest to plant biologists studying the interplay between dispersal, demography and genetics, as well as plant–animal interactions.

Incorporating exposure to pitch canker disease to support management decisions of Pinus pinaster Ait. in the face of climate change

Climate change is gravely affecting forest ecosystems, resulting in large distribution shifts as well as in increasing infection diseases and biological invasions. Accordingly, forest management requires an evaluation of exposure to climate change that should integrate both its abiotic and biotic components. Here we address the implications of climate change in an emerging disease by analysing both the host species (Pinus pinaster, Maritime pine) and the pathogen’s (Fusarium circinatum, pitch canker) environmental suitability i.e. estimating the host’s risk of habitat loss and the disease`s future environmental range. We constrained our study area to the Spanish Iberian Peninsula, where accurate climate and pitch canker occurrence databases were available. While P. pinaster is widely distributed across the study area, the disease has only been detected in its north-central and north-western edges. We fitted species distribution models for the current distribution of the conifer and the disease. Then, these models were projected into nine Global Climate Models and two different climatic scenarios which totalled to 18 different future climate predictions representative of 2050. Based on the level of agreement among them, we created future suitability maps for the pine and for the disease independently, which were then used to assess exposure of current populations of P. pinaster to abiotic and biotic effects of climate change. Almost the entire distribution of P. pinaster in the Spanish Iberian Peninsula will be subjected to abiotic exposure likely to be driven by the predicted increase in drought events in the future. Furthermore, we detected a reduction in exposure to pitch canker that will be concentrated along the north-western edge of the study area. Setting up breeding programs is recommended in highly exposed and productive populations, while silvicultural methods and monitoring should be applied in those less productive, but still exposed, populations.